Viral Vector-Based Techniques for Optogenetic Modulation In Vivo - Viral Vector Approaches in Neurobiology and Brain Diseases - page 291

15. Injection: virus withdrawal:

! Precaution: Any object that has contacted the virus

suspension is considered biohazardous material and
must be treated according to the existing regulations.
Such material can be placed in a designated biohaz-
ards vial containing ethanol (70 %) or H 2 O 2 .

Using the syringe, withdraw the virus suspension at a rate
of 20-50 nl/s. It is recommended to withdraw at least 1.5
times the exact volume needed in order to ensure suffi -
cient suspension.

●

16. Injection: preparation step:
! This stage is important to perform in order to assure that

no air remains at the needle tip after virus withdrawal.

● While syringe needle is still in the air, infuse until a drop
forms at the needle tip.
● Use Sugi tip to absorb the drop and discard into biohaz-
ard vial.
17. Injection: Infusion:

! At this stage, movement of the mouse, stereotax and

table should be minimized since any excessive vibra-
tion could cause movement of the needle inside the
brain tissue and leakage of virus through the gap
formed by this movement. Do not perform toe-pinch
refl ex tests until the syringe is retracted from the
brain and minimize any movements of the table .

Recommended: Before injecting, verify the position of
bregma and reset the stereotactic coordinates to this location
since the skull or needle may have shifted in previous steps.

●

Insert syringe through the drilled hole (while confi rming
correct AP and ML coordinates) and into the brain until
reaching the desired DV coordinate. This should be done
slowly once the brain is entered (at the rate of about
2 mm/min) for minimal tissue damage (Fig. 1 -bVI).

●

Wait about 5 min for brain tissue to adjust to needle at
coordinate.

●

Set infusion speed to 100 nl/min and begin infusion.

●

Monitor infusion: If fl uid is observed to accumulate at the
surface, stop injection, wait several minutes, and/or lower
the needle by several tens of microns before continuing
with infusion.

●

After infusion, wait 10 min for virus to diffuse away from
injection site.

●

Withdraw syringe slowly (~1 mm/min).

●